Non-invasive detection of fetal genetic traits

ABSTRACT

Blood plasma of pregnant women contains fetal and (generally &gt;90%) maternal circulatory extracellular DNA. Most of said fetal DNA contains ≦500 base pairs, said maternal DNA having a greater size. Separation of circulatory extracellular DNA of &lt;500 base pairs results in separation of fetal from maternal DNA. A fraction of a blood plasma or serum sample of a pregnant woman containing, due to size separation (e.g. by chromatography, density gradient centrifugation or nanotechnological methods), extracellular DNA substantially comprising ≦500 base pairs is useful for non-invasive detection of fetal genetic traits (including the fetal RhD gene in pregnancies at risk for HDN; fetal Y chromosome-specific sequences in pregnancies at risk for X chromosome-linked disorders; chromosomal aberrations; hereditary Mendelian genetic disorders and corresponding genetic markers; and traits decisive for paternity determination) by e.g. PCR, ligand chain reaction or probe hybridization techniques, or nucleic acid arrays.

RELATED APPLICATIONS

This patent application is a continuation of U.S. Patent Application No.10/964,726 filed on Oct. 15, 2004, entitled NON-INVASIVE DETECTION OFFETAL GENETIC TRAITS, naming Sinuhe Hahn, Wolfgang Holzgreve, BernhardZimmermann, and Ying Li as inventors, and designated by Attorney DocketNo. SEQ-5002-UT, which claims the benefit under 35 U.S.C. 119(a) ofEuropean Patent Application No. 03405742.2 filed on Oct. 16, 2003,entitled NON-INVASIVE DETECTION OF FETAL GENETIC TRAITS, naming SinuheHahn, Wolfgang Holzgreve, Bernhard Zimmermann, and Ying Li as inventorsand designated by Attorney Docket No. SEQ-5002-EP. The entirety of eachof these patent applications is incorporated herein by reference.

BACKGROUND OF THE INVENTION

The presence of circulatory extracellular DNA in the peripheral blood isa well established phenomenon. In this context, it has been shown thatin the case of a pregnant woman extracellular fetal DNA is present inthe maternal circulation and can be detected in maternal plasma orserum. Studies have shown that this circulatory fetal genetic materialcan be used for the very reliable determination, e.g. by PCR (polymerasechain reaction) technology, of fetal genetic loci which are completelyabsent from the maternal genome. Examples of such fetal genetic loci arethe fetal RhD gene in pregnancies at risk for HDN (hemolytic disease ofthe fetus and newborn) or fetal Y chromosome-specific sequences inpregnancies at risk for an X chromosome-linked disorder e.g. hemophiliaor fragile X syndrome.

The determination of other, more complex fetal genetic loci (e.g.chromosomal aberrations such as aneuploidies or chromosomal aberrationsassociated with Down's syndrome, or hereditary Mendelian geneticdisorders and, respectively, genetic markers associated therewith, suchas single gene disorders, e.g. cystic fibrosis or thehemoglobinopathies) is, however, more problematic. The reason for thisdifficulty is that the major proportion (generally >90%) of theextracellular DNA in the maternal circulation is derived from themother. This vast bulk of maternal circulatory extracellular DNA rendersit difficult, if not impossible, to determine fetal genetic alternationssuch as those involved in chromosomal aberrations (e.g. aneuploidies) orhereditary Mendelian genetic disorders (e.g. cystic fibrosis or thehemoglobinopathies) from the small amount of circulatory extracellularfetal DNA.

SUMMARY OF THE INVENTION

An examination of circulatory extracellular fetal DNA and circulatoryextracellular maternal DNA in maternal plasma has now shown that,surprisingly, the majority of the circulatory extracellular fetal DNAhas a relatively small size of approximately 500 base pairs or less,whereas the majority of circulatory extracellular maternal DNA inmaternal plasma has a size greater than approximately 500 base pairs.Indeed, in certain instances the circulatory DNA material which issmaller than approximately 500 base pairs appears to be almost entirelyfetal. Circulatory extracellular fetal DNA in the maternal circulationhas thus been found to be smaller in size (approximately 500 base pairsor less) than circulatory extracellular maternal DNA (greater thanapproximately 500 base pairs).

This surprising finding forms the basis of the present inventionaccording to which separation of circulatory extracellular DNA fragmentswhich are smaller than approximately 500 base pairs provides apossibility to enrich for fetal DNA sequences from the vast bulk ofcirculatory extracellular maternal DNA.

This selective enrichment, which is based on size discrimination ofcirculatory DNA fragments of approximately 500 base pairs or less, leadsto a fraction which is largely constituted by fetal extracellular DNA.This permits the analysis of fetal genetic traits including thoseinvolved in chromosomal aberrations (e.g. aneuploidies or chromosomalaberrations associated with Down's syndrome) or hereditary Mendeliangenetic disorders and, respectively, genetic markers associatedtherewith (e.g. single gene disorders such as cystic fibrosis or thehemoglobinopathies), the determination of which had, as mentioned above,so far proved difficult, if not impossible. Size separation ofextracellular fetal DNA in the maternal circulation thus facilitates thenon-invasive detection of fetal genetic traits, including paternallyinherited polymorphisms which permit paternity testing.

Clinical Chemistry, 1999, Vol. 45(9), pages 1570-1572 and The Australian& New Zealand

Journal of Obstetrics & Gynaecology, February 2003 (O.sub.2-2003), Vol.43(1), pages 10-15 describe a sample of blood plasma of a pregnant womanin which extracellular fetal DNA of less than 500 base pairs is enrichedby PCR, is separated by gel electrophoresis and fetal male DNA (fetalY-chromosome-specific sequence) is detected.

The present invention provides: a fraction of a sample of the bloodplasma or serum (which preferably is substantially cell-free) of apregnant woman in which, as the result of said sample having beensubmitted to a size separation, the extracellular DNA present thereinsubstantially consists of DNA comprising 500 base pairs or less; the useof such sample-fraction for the non-invasive detection of fetal genetictraits; and a process for performing non-invasive detection of fetalgenetic traits which comprises subjecting a sample of the blood plasmaor serum of a pregnant woman to a size separation so as to obtain afraction of said sample in which the extracellular DNA present thereinsubstantially consists of DNA comprising 500 base pairs or less, anddetermining in said sample-fraction the fetal genetic trait(s) to bedetected.

Said serum or plasma sample is preferably substantially cell-free, andthis can be achieved by known methods such as, for example,centrifugation or sterile filtration.

The size separation of the extracellular DNA in said serum or plasmasample can be brought about by a variety of methods, including but notlimited to: chromatography or electrophoresis such as chromatography onagarose or polyacrylamide gels, ion-pair reversed-phase high performanceliquid chromatography (IP RP HPLC, see Hecker K H, Green S M, KobayashiK, J. Biochem. Biophys. Methods 2000 Nov. 20; 46(1-2): 83-93), capillaryelectrophoresis in a self-coating, low-viscosity polymer matrix (see DuM, Flanagan J H Jr, Lin B, Ma Y, Electrophoresis 2003 September; 24(18): 3147-53), selective extraction in microfabricated electrophoresisdevices (see Lin R, Burke D T, Burn M A, J. Chromatogr. A. 2003 Aug. 29;1010(2): 255-68), microchip electrophoresis on reduced viscosity polymermatrices (see Xu F, Jabasini M, Liu S, Baba Y, Analyst. 2003 June;128(6): 589-92), adsorptive membrane chromatography (see Teeters M A,Conrardy S E, Thomas B L, Root T W, Lightfoot E N, J. Chromatogr. A.2003 Mar. 7; 989(1): 165-73) and the like; density gradientcentrifugation (see Raptis L, Menard H A, J. Clin. Invest. 1980December; 66(6): 1391-9); and methods utilising nanotechnological meanssuch as microfabricated entropic trap arrays (see Han J, Craighead H G,Analytical Chemistry, Vol. 74, No. 2, Jan. 15, 2002) and the like.

The sample-fraction thus obtained not only permits the subsequentdetermination of fetal genetic traits which had already been easilydetectable in a conventional manner such as the fetal RhD gene inpregnancies at risk for HDN (hemolytic disease of the fetus and thenewborn), or fetal Y chromosome-specific sequences in pregnancies atrisk for an X chromosome-linked disorder such as hemophilia, fragile Xsyndrome or the like, but also the determination of other, more complexfetal genetic loci, including but not limited to: chromosomalaberrations (e.g aneuploidies or Down's syndrome) or hereditaryMendelian genetic disorders and, respectively, genetic markersassociated therewith (e.g. single gene disorders such as cystic fibrosisor the hemoglobinopathies); and fetal genetic traits which may bedecisive when paternity is to be determined.

Such determination of fetal genetic traits can be effected by methodssuch as, for example, PCR (polymerase chain reaction) technology, ligasechain reaction, probe hybridization techniques, nucleic acid arrays(so-called “DNA chips”) and the like.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following Examples further illustrate the invention but are not tobe construed as limitating its scope in any way.

EXAMPLE 1 Detection of Male Fetal DNA in Maternal Plasma by Real-TimeQuantitative Polymerase Chain Reaction (PCR) After Size Fractionation ofDNA by Agarose Gel Electrophoresis Materials and Methods

Subjects and Sample Processing

Seven women pregnant in the third trimester with a male fetus wererecruited for this study. 16-18 ml blood samples were collected intoEDTA tubes. 6-9 ml of plasma were obtained after centrifugation at 1600g for 10 minutes and a second centrifugation of the supernatant at 16000g for 10 minutes.

DNA Isolation

DNA from 5-7 ml plasma was extracted using the QIAgen Maxi kit,according to the manufacturers' protocol. DNA was eluted in a volume of1.5 ml.

DNA Precipitation

1. To the plasma DNA were added: 1/10 volume NaAc (3M, pH 5.2), 2volumes absolute ethanol, MgCl₂ to a final concentration of 0.01 M andGlycogen to a final concentration of 50 μg/ml. The solution wasthoroughly mixed by vortexing.

2. The solution was stored overnight at −70° C.

3. The DNA was recovered by centrifugation at 20000 g for 30 minutes at4° C.

4. The supernatant was carefully removed and the pellet washed with 500μl 70% ethanol.

5. The pellet was air dried and dissolved in 35 μl distilled water.

DNA Separation

1. A 1% agarose Gel (Invitrogen, Cat No: 15510-027) was prepared for DNAelectrophoresis.

2. 28 μl DNA solution were loaded on the gel.

3. The gel was electrophoresed at 80 Volt for 1 hour.

4. The Gel was cut into pieces corresponding to specific DNA sizesaccording to the DNA size markers (New England Biolabs, 100 bp ladderand Lamda Hind III digest). The DNA sizes contained by the specific gelfragments were: 90-300 bases, 300-500 bases, 500-1000 bases, 1.0-1.5kilobases (“kb”), 1.5-23 kb and >23 kb.

5. The DNA was purified from the agarose gel pieces using the QIAEX IIGel Extraction kit (Qiagen, Cat No. 20021) and eluted in 35 μl Tris-HCl(pH 8.0, 10 mM).

Real-Time PCR

Sequences from the Y chromosome (SRY) and from chromosome 12 (GAPDHgene) were amplified with the Applied Biosystems (ABI) 7000 SequenceDetection System by real-time quantitative PCR to quantify amounts offetal and total DNA in the size-separated fractions. The TaqMan systemfor SRY consisted of the amplification primers SRY_Fwd: TCC TCA AAA GAAACC GTG CAT (SEQ ID NO: 1) and SRY Rev: AGA TTA ATG GTT GCT AAG GAC TGGAT (SEQ ID NO: 2) and a FAM labeled TaqMan MGB (Minor Groove Binder)probe SRY_MGB: TCC CCA CAA CCT CTT (SEQ ID NO: 3). The TaqMan System forthe GAPDH gene consisted of the following primers and probe: GAPDH_Fwd:CCC CAC ACA CAT GCA CTT ACC (SEQ ID NO: 4), GAPDH_Rev: CCT AGT CCC AGGGCT TTG ATT (SEQ ID NO: 5) and GAPDH_MGB: TAG GAA GGA CAG GCA AC (SEQ IDNO: 6).

TaqMan amplification reactions were set up in a total reaction volume of25 μl, containing 6 μl of the sample DNA solution, 300 nM of each primer(HPLC purified, Mycrosynth, Switzerland) and 200 nM of each probe (ABI)at 1× concentration of the Universal PCR reaction mix (ABI). Each samplewas analyzed in duplicate for each of the two amplification systems. Astandard curve containing known amounts of genomic DNA was run inparallel with each analysis.

Thermal cycling was performed according to the following protocol: aninitial incubation at 50° C. for 2 minutes to permit Amp Erase activity,10 minutes at 95° C. for activation of AmpliTaq Gold, and 40 cycles of 1minute at 60° C. and 15 seconds at 95° C. Amplification data collectedby the 7000 Sequence Detection System was quantified using the slope ofthe standard curve as calculated by the sequence detection software andthe results of a standard DNA solution used in the dilution curve withsimilar DNA copy numbers as the sample reactions as a reference samplefor copy number calculations.

Results

Table 1 shows that in the five pregnancies examined, DNA fragmentsoriginating from the fetus were almost completely of sizes smaller than500 base pairs with around 70% being of fetal origin for sizes smallerthan 300 bases.

These results demonstrate that free DNA of fetal origin circulating inthe maternal circulation can be specifically enriched by size separationof the total free DNA in the maternal blood.

Depending on the downstream application the DNA size chosen for theenrichment of fetal DNA will be smaller than 300 or smaller than 500bases.

TABLE 1 % of fetal DNA % of maternal DNA Size of DNA in each fragment ineach fragment <0.3 kb  73.2 (22.22-87.06)   26.8 (12.94-77.78) 0.3-0.5kb 18.95 (6.43-31.42)  81.05 (68.58-93.57) 0.5-1 kb 2.81 (0.00-7.75)97.19 (92.25-100) 1.0-1.5 kB  0.00 (0.00-12.50)  100 (87.5-100) 1.5-23kb 0.00 (0.00-8.40) 100 (100-100)

The abbreviation “kb” appearing in the first column of this table standsfor 1000 base pairs, and the figures given in its second and the thirdcolumn are the median values of the percentages and, in brackets, theranges.

EXAMPLE 2 Detection of Fetal DNA After Agarose Gel Electrophoresis byPolymerase Chain Reaction (PCR) of Microsatellite Markers, also Called“Short Tandem Repeats” (STRs) Materials and Methods

Subjects and Samples

18 ml blood samples from pregnant women and 9 ml blood from theirpartners were collected into EDTA tubes and plasma separated bycentrifugation as described in Example 1. The maternal buffy coat (i.e.the white colored top layer of the cell pellet obtained after the firstcentrifugation of 1600 g for 10 min.) was washed twice with PBS.

DNA Isolation

DNA from the plasma was extracted using a modification of the High PureDNA template kit from Roche, the whole sample was passed through thefilter usually used for 200 μl using a vacuum. The DNA was eluted in avolume of 50 μl elution buffer. Paternal DNA was extracted from 400 μlpaternal whole blood, using the High Pure DNA template kit, and elutedinto 100 μl. Maternal DNA was isolated from the buffy coat, using theHigh Pure DNA template kit, and eluted into 100 μl.

DNA Separation

The DNA was size-separated by electrophoresis on an agarose gel andpurified as described in Example 1.

PCR Specific for Short Tandem Repeats

From the fraction of sizes smaller than 500 bases, sequences fromtetranucleotide repeat markers on Chromosome 21 were amplified in amultiplex PCR reaction as described in Li et al. Clinical Chemistry 49,No. 4, 2003. Because of the low concentration of plasma DNA, the fetalDNA in maternal plasma was examined by using a semi-nested PCR protocol.

The maternal and paternal pairs were genotyped using total genomic DNAto monitor microsatellite markers on chromosome 21.

The STR markers used were:

-   -   D211 S11;    -   D21S1270;    -   D21S1432; and    -   D21S1435

The resulting DNA fragments were then size separated by capillaryelectrophoresis on a sequencer, and the peak areas representing eachallele for a specific marker were measured by the software.

Results

TABLE 2 Detection of fetal alleles specific for the microsatellitemarker (Short Tandem Repeat) D21S11 on chromosome 21 Maternal Fetalalleles alleles detected detected (D21S11) (D21S11) Maternal genomic 232bp N/A DNA 234 bp Total extracellulear 232 bp No fetal DNA (unseparated)234 bp alleles detectable Size-separated 232 bp 228 bp extracellular DNA234 bp 232 bp (<300 bp) Size-separated 232 bp 228 bp extracellular DNA234 bp 232 bp (300-500 bp)

Only in the size-separated fractions (<300 by and 300-500 bp) could thefetal alleles for D21S11 be detected, namely the paternally inherited228 by allele and the maternally inherited 232 by allele, i.e., oneallele from each parent.

Discussion

Analysis of the STR fragments can allow for the detection of paternalalleles that are distinct in length from the maternal repeat sequences,and by calculating the ratios between the peak areas it can be possibleto identify patterns that are not consistent with a normal fetalkaryotype. The identification of paternal allele sizes of STRs in thematernal circulation can allow the detection of certain chromosomalaberrations non-invasively. Also paternity testing can be accomplishedprenatal in a non-invasive manner.

1. Isolated deoxyribonucleic acid (DNA), consisting of: DNA fragments ofsubstantially 500 base pairs or less comprising a fraction of DNA fromblood plasma of a pregnant female largely constituted of circulatingextracellular fetal DNA, which DNA fragments are not in association witha gel, and which circulating extracellular fetal DNA in the isolated DNAis enriched relative to circulating extracellular DNA in the bloodplasma of the pregnant female.
 2. The isolated DNA of claim 1, whereinthe DNA fragments are substantially 300 base pairs or less.
 3. Theisolated DNA of claim 2, wherein the DNA fragments are at least about70% circulating extracellular fetal DNA.
 4. The isolated DNA of claim 1,wherein the fetal DNA comprises an allele different than an allele inthe maternal DNA.
 5. The isolated DNA of claim 1, wherein the fetal DNAcomprises a chromosome aneuploidy.
 6. The isolated DNA of claim 1,wherein the fetal nucleic acid comprises a paternally inheritedpolymorphism not present in a maternally inherited sequence.
 7. Thecomposition of claim 6, wherein the paternally inherited polymorphism isof a length different than in the maternally inherited sequence.
 8. Theisolated DNA of claim 1, wherein the DNA fragments comprise DNA that canamplify DNA fragments in the isolated DNA.
 9. The isolated DNA of claim8, wherein the DNA fragments comprise a DNA primer that can amplify DNAfragments in the isolated DNA.
 10. The isolated DNA of claim 1, whereinthe DNA fragments are in solution.
 11. Isolated deoxyribonucleic acid(DNA), consisting of: DNA fragments of substantially 500 base pairs orless comprising a fraction of DNA from blood plasma of a pregnant femalelargely constituted of circulating extracellular fetal DNA, which DNAfragments are not in association with a gel, which circulatingextracellular fetal DNA in the isolated DNA is enriched relative tocirculating extracellular DNA in the blood plasma of the pregnantfemale, and which isolated DNA is prepared by a process comprising: (a)extracting circulating extracellular DNA from the plasma from thepregnant female, thereby providing extracted DNA; and (b) separating andisolating the extracted DNA by size, thereby providing separated andisolated DNA; whereby the separated and isolated DNA is enriched for thecirculating extracellular fetal DNA.
 12. The isolated DNA of claim 11,wherein the DNA fragments are substantially 300 base pairs or less. 13.The isolated DNA of claim 12, wherein the DNA fragments are at leastabout 70% circulating extracellular fetal DNA.
 14. The isolated DNA ofclaim 11, wherein the fetal DNA comprises an allele different than anallele in the maternal DNA.
 15. The isolated DNA of claim 11, whereinthe fetal DNA comprises a chromosome aneuploidy.
 16. The isolated DNA ofclaim 11, wherein the DNA fragments comprise DNA that can amplify DNAfragments in the isolated DNA.
 17. The isolated DNA of claim 16, whereinthe DNA fragments comprise a DNA primer that can amplify DNA fragmentsin the isolated DNA.
 18. The isolated DNA of claim 11, wherein the DNAfragments are in solution.
 19. The isolated DNA of claim 11, whereinseparating the DNA in step (b) comprises electrophoresis.
 20. Theisolated DNA of claim 11, wherein separating the DNA in step (b)comprises chromatography.